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Vollständiger Abdruck der von der Fakultät für Medizin der Technischen
Universität München zur Erlangung des akademischen Grades eines Doktors der
Medizin (Dr. med.) genehmigten Dissertation.
Vorsitzender: Prof. Dr. Jürgen Schlegel
Prüfende der Dissertation:
3. apl. Prof. Dr. Lutz Renders
Die Dissertation wurde am 03.08.2020 bei der Technischen Universität München
eingereicht und durch die Fakultät für Medizin am 16.02.2021 angenommen.
In Gedenken an meinen Vater, Dr. med. Hans Harald Riedhammer.
Everything is going to be fine in the end. If it's not fine it's not the end.
Oscar Wilde
ABSTRACT
Introduction: Hereditary kidney diseases affect about one in ten adults with chronic
kidney disease (CKD) and about two-thirds of patients with CKD-onset under the age of
25 years. Hence, they pose a considerable burden of disease. All parts of the intricate
organ that is the kidney and urinary tract can be altered and hereditary nephropathies are
therefore clinically and genetically vastly heterogeneous. Exome sequencing (ES), that
is, the analysis of the protein-coding regions of the human genome, is able to address this
genetic heterogeneity. Aim of this thesis: Evaluation of ES in 260 index cases with a
clinically presumed hereditary nephropathy with emphasis on the detection of
phenocopies (clinical tentative diagnosis is different from genetic diagnosis), the
prioritization of novel disease-associated genes (“candidate genes”), and the statistical
analysis of the cohort to improve clinical decision-making. Study design: Cross-sectional
study. Methods: ES in 260 genetically unsolved index cases recruited between October
2015 and February 2019. Results: 77 of 260 cases could be genetically solved (a
diagnostic yield of 30%). In 12 of 77 solved cases (16%), a phenocopy was identified. In
8 of 260 cases (3%), a candidate gene could be prioritized. There were significant
differences in Alport syndrome (AS) versus thin basement membrane nephropathy
(TBMN), the two poles of disease severity of type-IV-collagen-related nephropathy:
Diagnostic yield was significantly higher in AS than in TBMN (65% vs. 28%, p = 0.01).
Median age at first manifestation was significantly lower in AS than in TBMN (5.5 years
[3.0–9.0] vs. 16.0 years [5.0–32.3], p = 0.001). There were no extrarenal manifestations
in TBMN cases, compared to 28% in AS cases (p = 0.01). A family history was less
commonly reported in TBMN cases than in AS cases (39% vs. 78%, p = 0.006). For the
total cohort, clinical predictors of a solved case were positive family history (odds ratio
[OR] 6.61 [95% confidence interval 3.28–13.35], p < 0.001), an extrarenal manifestation
(OR 3.21 [1.58–6.54], p = 0.01) and – with a borderline significance – younger age at
first manifestation (OR 0.97 [0.93–1.00], p = 0.048). Discussion: This thesis shows the
utility of ES in hereditary nephropathies by the identification of phenocopies, which have
major implications for disease management and prognosis, and of novel disease-
associated genes. Furthermore, the results of this thesis guide the genetic work-up of
patients by presenting statistical evidence for predictors of a positive genetic result and
for delineating the disease spectrum of type-IV-collagen-related nephropathy.
5
ACKNOWLEDGMENTS
I would like to thank my doctoral supervisor, PD Dr. Julia Hoefele, for her excellent
support. Additionally, many thanks to Univ.-Prof. Dr. Thomas Meitinger, the head of the
Institute of Human Genetics, Klinikum rechts der Isar, Technical University of Munich,
Munich, Germany, for filling me with enthusiasm for the field of human genetics.
My family has been a beacon of support – my mother, Margit Riedhammer; my sister,
Anna-Katharina Hiemer; my brother-in-law, Daniel Hiemer; and my girlfriend, Johanna
Greiner. All my love to them: I would not have completed this thesis without them.
I would further like to thank the medical technical assistants at the Institute of Human
Genetics, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany,
and at the Institute of Human Genetics and the Next-Generation Sequencing Core Facility
at the Helmholtz Zentrum München – German Research Center for Environmental
Health, Neuherberg, Germany. Further thanks to Dr. Matthias Braunisch, Dr. Bettina
Lorenz-Depiereux, and Dr. Matias Wagner for proofreading of the manuscript.
Thank you to Kayla Friedman and Malcolm Morgan of the Centre for Sustainable
Development, University of Cambridge, United Kingdom, for producing the Microsoft
Word thesis template used to produce this document.
Finally, I thank all referring clinicians and collaboration partners and, last but not least,
the index patients and their legal guardians for participating in this study.
6
ONLINE RESOURCES ..................................................................................................... 23
1 INTRODUCTION ...................................................................................................... 25
1.1.2 Alport syndrome (AS) .................................................................................... 31
1.1.3 Congenital anomalies of the kidney and urinary tract (CAKUT) .................. 35
1.1.4 Ciliopathies .................................................................................................... 40
syndrome (FSGS/SRNS) ......................................................................................... 47
1.1.7.1 Tubulopathies .................................................................................................................................... 54
1.1.7.3 Mitochondrial disorders .................................................................................................................... 56
1.2 EXOME SEQUENCING .............................................................................................. 58
1.2.1 Exome sequencing in general, its utility and limitations ............................... 58
1.2.2 Exome sequencing in hereditary nephropathies ............................................. 63
1.2.3 Comprehensive genetic testing for the detection of phenocopies in hereditary
nephropathies .......................................................................................................... 66
2 AIM OF THIS THESIS ............................................................................................. 70
3 PATIENT COHORT, MATERIAL, AND METHODS ......................................... 71
3.1 PATIENT COHORT ................................................................................................... 71
3.1.2 Phenotype ascertainment ................................................................................ 73
3.2 SAMPLE PROCESSING, POLYMERASE CHAIN REACTION, AND SANGER SEQUENCING 77
3.2.1 DNA isolation ................................................................................................ 77
3.2.2 DNA quantification and quality check ........................................................... 78
3.2.3 Polymerase chain reaction ............................................................................. 79
3.2.3.1 Basic method ..................................................................................................................................... 79
3.2.3.4 PCR purification ............................................................................................................................... 81
3.2.4 Sanger sequencing .......................................................................................... 81
3.2.4.1 Basic method ..................................................................................................................................... 81
3.2.4.3 Sequencing ........................................................................................................................................ 83
3.4 STATISTICAL ANALYSIS AND GRAPHICAL VISUALIZATION ...................................... 92
4 RESULTS ................................................................................................................... 93
4.1 EXOME SEQUENCING IN A COHORT OF 260 GENETICALLY UNSOLVED INDEX CASES
WITH A CLINICALLY PRESUMED HEREDITARY NEPHROPATHY ....................................... 93
4.1.1 Cohort description .......................................................................................... 93
4.1.2 Diagnosis of a hereditary kidney disease by exome sequencing ................. 101
4.2 DIFFERENCES IN TYPE-IV-COLLAGEN-RELATED NEPHROPATHY CLASSIFIED AS
ALPORT SYNDROME VERSUS THIN BASEMENT MEMBRANE NEPHROPATHY ................. 104
4.3 DETECTION OF PHENOCOPIES AND RECATEGORIZATION OF DISEASE BY EXOME
SEQUENCING............................................................................................................... 104
4.3.2 Phenocopy detection rate of targeted NGS panels ....................................... 111
4.4 EXOME SEQUENCING TO PRIORITIZE NOVEL HEREDITARY-KIDNEY-DISEASE-
ASSOCIATED GENES .................................................................................................... 111
5 DISCUSSION ........................................................................................................... 116
8
5.1 DIAGNOSTIC YIELD IN INDEX PATIENTS WITH A CLINICALLY PRESUMED HEREDITARY
NEPHROPATHY EXAMINED BY EXOME SEQUENCING .................................................... 116
5.2 TYPE-IV-COLLAGEN-RELATED NEPHROPATHY – ALPORT SYNDROME VS. THIN
BASEMENT MEMBRANE NEPHROPATHY ....................................................................... 119
IMPLICATIONS OF THE RECATEGORIZATION OF DISEASE ............................................. 122
5.4 NOVEL HEREDITARY-KIDNEY-DISEASE-ASSOCIATED GENES ................................. 126
5.5 WHOM TO TEST? PREDICTORS OF A POSITIVE GENETIC TEST ................................. 129
5.6 LIMITATIONS ........................................................................................................ 131
6 REFERENCES ......................................................................................................... 135
7 APPENDICES .......................................................................................................... 174
7.3 PUBLICATIONS PERTINENT TO THE THESIS ............................................................ 177
7.4 FURTHER PUBLICATIONS ...................................................................................... 177
7.5 PRESENTATIONS AT CONFERENCES ....................................................................... 180
7.6 URL/DOI FOR ONLINE SUPPLEMENTARY TABLE (FEATURING ALL INDEX CASES WITH
DETAILED PHENOTYPIC AND GENOTYPIC INFORMATION) ............................................ 182
9
Glossary
Adapted from Strachan & Read, 2018, if not specifically indicated.
3’ end End of a DNA/RNA strand at the third carbon of
the sugar-ring of a nucleic acid
5’ end End of a DNA/RNA strand at the fifth carbon of the
sugar-ring of a nucleic acid
Allele Different types of the same gene
Allele frequency Frequency of an allele at a certain locus in a
population
Allelic heterogeneity Different variants in the same gene leading to the
same phenotype in different individuals
Amino acid Molecules that constitute proteins (there are 20
proteinogenic amino acids in the standard genetic
code)
Amplification Increase in the amount of a DNA sequence (e.g., by
PCR)
nucleic acids
of an individual (e.g., phenotype)
Autosome Chromosomes that are not sex chromosomes (i.e.,
chromosomes 1–22).
nonhomologous chromosomes in which there is no
loss of genetic material
Base complementarity Association of two bases on opposite strands of
double-stranded nucleic acids (A with T [in DNA]
or U [in RNA], G with C)
10
(pairing of a purine base with a pyrimidine base by
a hydrogen bond)
RNA by reverse transcriptase
containing genetic information (genes)
Codon A triplet of nucleotides in RNA translated into an
amino acid (or start/stop signal for translation)
Common variant Variant with an allele frequency > 5.0% in a
population
Compound heterozygote An individual with two different alleles at the same
locus
Consanguinity When mating partners have a known shared
ancestor (e.g., first cousins)
Conserved sequence A DNA (or amino acid) sequence that is the same
in multiple species
kilobase pairs (older definition; Feuk et al., 2006;
Nowakowska, 2017)
repeats- (CRISPR-) associated – part of the
adaptive immune system of prokaryotes, utilized
for genome editing
Cytogenetics A branch of genetics engaged in the study of
chromosomes
11
De novo variant A variant identified in an individual but not in the
biological parents of this individual
Denaturation Disassembly of double strands of nucleic acids into
single strands (also, the destruction of protein
structure)
(normal state of human somatic cells)
DNA polymerase Enzymes that elongate DNA at the 3’ end
Dominant A trait manifesting in heterozygotes
Dominant negative effect Heterozygous variant leading to an altered protein,
which affects the function of the non-altered protein
Epigenetic Inherited phenotype not caused by an altered DNA
sequence
exome-sequencing data of 60,706 unrelated
individuals; Lek et al., 2016)
Exome The entirety of exons of the genome
Exon Parts of a gene present in spliced RNA
Expressivity Variable range of a phenotype caused by a
particular genotype
Frameshift variant A variant leading to an alteration of the reading
frame of mRNA (due to insertion/deletion of
nucleotides by a number which cannot be divided
by three)
functional noncoding RNA)
functional noncoding RNA)
12
Gene knockout Abrogation of expression of a certain gene in a
cell/organism (e.g., mouse)
Genetic redundancy The loss of one gene product can be compensated
by the gene product of a different gene
Genotype List of alleles of a certain individual at one or more
loci
Genotype-phenotype correlation Prediction of a phenotype from a given genotype
gnomAD Genome Aggregation Database (database of
125,748 exome and 15,708 genome sequences from
unrelated individuals [v.2.1.1]; Karczewski et al.,
2020)
Haploid One copy of every chromosome is present (e.g., in
sperm and egg cells)
Haploinsufficiency If there is a loss of one gene product (e.g., a
heterozygous nonsense variant), in a
haploinsufficiency locus, a phenotype will occur
Haplotype Cluster of linked alleles on a single chromosome (in
linkage disequilibrium)
Hardy-Weinberg principle Relation of allele and genotype frequencies in a
population
Hemizygous One copy of DNA/a gene in a diploid cell (e.g., X-
chromosomal genes in males)
variant
Heterozygote An individual who has two different alleles at a
certain locus
Homozygote The same alleles at a certain locus
13
Hypomorphic variant A variant leading to a mild phenotype (altered gene
product has a residual function/expression; Wilkie,
1994)
reference genotype panels of known haplotypes
(used in genome-wide association studies)
In cis Variants in a gene, which are in cis, are on the same
chromosome (= monoallelic;
Indel Insertion or deletion of bases
In trans Variants in a gene, which are in trans, are on
different chromosomes (= biallelic;
Intron Parts of a DNA sequence removed by the splicing
of RNA
Karyotype The set of chromosomes of a cell or an individual
Linkage disequilibrium Alleles at linked loci with limited recombination
Locus Defined position of a gene/DNA sequence within
the genome
different genes/loci
Loss-of-function variant A variant leading to a loss of gene product function
(e.g., a nonsense variant)
inheritable by Mendelian laws (i.e., a monogenic
disease)
14
translation
Missense variant Variant leading to an exchange of amino acids in
the respective protein
Monosomy The condition of having a single copy of a
chromosome (e.g., monosomy X)
Motif A distinct sequence, typically in a protein,
important for structural/functional properties
several factors (genetic, epigenetic, environmental)
Mutation The event of a DNA sequence change (also used to
describe the result of the change, e.g., missense
mutation)
Nonsense variant A variant leading to a premature termination codon
Nonsense-mediated mRNA decay Pathway of mRNA surveillance degrading
transcripts with premature termination codons (e.g.,
due to a nonsense variant). Nonsense-mediated
mRNA decay (NMD) of a transcript is expected
(but not always occurring) if a there is a stop in
translation more than 50 nucleotides before the last
exon–exon junction (Lambert et al., 2020).
Nucleic acid DNA/RNA
(ribose/deoxyribose)
15
phosphate)
Open reading frame DNA sequence without a stop codon in a particular
reading frame
Paired-end sequencing Sequencing both ends of a DNA fragment and
comparing the nucleotide number between these
sequences to the reference genome in order to
detect rearrangements such as deletions or
duplications
Penetrance The proportion with which a genotype results in a
phenotype
Pleiotropy Variants in a gene lead to multiple phenotypic traits
Polygenic A phenotype is the result of several genetic loci
acting together
speaking, two or more variants in a population at a
frequency too high to be due to repeated mutations)
Positive selection A certain genotype is favored in evolution
Protein domain A particular structure/functional unit within a
protein
coding gene, but without function
Purine bases Adenine and guanine
Pyrimidine bases Cytosine, thymine, and uracil
Rare variant A variant with an allele frequency < 1.0%
16
sequence relating to a specific DNA fragment (the
number of reads is called “read depth”)
Recessive A phenotype only manifesting if both copies of a
gene are affected (homozygous/compound-
Segregation In terms of pedigrees, the likelihood of inheriting a
trait/phenotype from a parent.
sequences
change
connecting of exons
Stop codon UAA, UAG, UGA in mRNA; leads to the
termination of protein translation
Synonymous variant A nucleotide change not leading to an amino acid
change
transcription
chromosome (e.g., trisomy 21)
Truncating variant Variant which leads to a shortened gene product
(also called protein-truncating variant; Rivas et al.,
2015)
Untranslated regions Parts of the mRNA at the 5’ and 3’ end not
translated into a protein that have important
regulatory functions
17
Variant of uncertain significance A variant that cannot be deemed (likely) benign or
(likely) pathogenic given current knowledge
X-inactivation Inactivation of all but one X chromosome in cells
with more than one X chromosome by epigenetic
mechanisms (in females; also called lyonization,
after geneticist Mary Lyon)
aa Amino acid
Kingdom)
Genomics
ADPKD Autosomal dominant polycystic kidney disease
ADTKD Autosomal dominant tubulointerstitial kidney
disease
AR Autosomal recessive
ARPKD Autosomal recessive polycystic kidney disease
AS Alport syndrome
tract
repeats
EDTA Ethylenediaminetetraacetic acid
EVS Exome Variant Server
FGGS Focal global glomerulosclerosis
FSGS Focal segmental glomerulosclerosis
GBM Glomerular basement membrane
GFR Glomerular filtration rate
Resources”)
Resources”)
20
Committee
IgAN IgA nephropathy
kb Kilobase pairs
KO Knockout
fraction
Resources”)
and stroke-like episodes
Resources”)
MRI Magnetic resonance imaging
“Online Resources”)
TAL Thick ascending limb of the loop of Henle
TBMN Thin basement membrane nephropathy
TMA Thrombotic microangiopathy
malformations, C – Cardiovascular anomalies, T –
Tracheoesophageal fistula, E – Esophageal atresia,
R – Renal and/or radial anomalies, L – Limb defects
VNTR Variable-number tandem repeats
XL X-linked
DECIPHER https://decipher.sanger.ac.uk/ (database of genomic
variation relating to human disease, especially
CNVs)
researchers interested in the same gene)
gnomAD https://gnomad.broadinstitute.org/ (database
genome sequencing projects; v.2.1.1 was used for
this thesis)
mouse models)
variant nomenclature)
and associated diseases)
PubMed https://pubmed.ncbi.nlm.nih.gov/ (database of
sequences)
24
hosted by the University of California, Santa Cruz,
CA, United States of America [UCSC])
UniProt https://www.uniprot.org/ (database of protein
sequences)
25
1 INTRODUCTION
Although each hereditary nephropathy (= hereditary kidney disease) on its own is usually
rare (affecting less than 1 in 2,000 people), hereditary kidney diseases in total affect
nearly 10% of adults with chronic kidney disease (CKD) and more than 70% of CKD
cases with onset below the age of 25 years. Therefore, hereditary nephropathies pose a
substantial burden of disease (Devuyst et al., 2014; Groopman et al., 2019; Vivante &
Hildebrandt, 2016). This thesis examines the application of exome sequencing (ES), that
is, the sequencing of the exonic (protein-coding) regions of the human genome, in 260
genetically unsolved index patients with a clinically presumed hereditary nephropathy
across all major disease groups (recruitment criteria are defined in Section 3.1). The
introduction describes the most important hereditary kidney diseases/disease entities and
the application of next-generation sequencing, specifically exome sequencing, in
hereditary nephropathies. The aim of this thesis is addressed after the introduction, in
Chapter 2.
Of note, the terms “monogenic,” “familial,” and “hereditary,” used interchangeably in
this thesis, denote a single-gene/copy number variant (CNV) cause for a disease
inheritable by Mendelian laws. In this thesis, the neutral term “variant” instead of
“mutation” for a change in human nucleotide sequence is used, as is preferred by the
American College of Medical Genetics and Genomics (ACMG; Richards et al., 2015).
Furthermore, wherever the expression “causative variant” is employed, this includes both
pathogenic and likely pathogenic variants/CNVs according to the ACMG criteria for
sequence variant/CNV interpretation (and amendments) with a fitting genotype (Section
Introduction
26
3.3.2; Richards et al., 2015; Riggs et al., 2020). Additionally, only gene names approved
by the Human Genome Organisation Gene Nomenclature Committee (HGNC) are used
(https://www.genenames.org/). The term “exome sequencing” instead of “whole exome
sequencing” (WES) is employed. Although common in the literature, WES is a) a
tautology and b) misleading, as (short-read-based) exome sequencing is not able to
capture the entirety of the protein-coding regions of the human genome, the “whole
exome,” as there are regions/genes with (partially) insufficient coverage or mapping
(assignment of sequenced DNA to a reference genome), for example, due to pseudogenes
or homologous regions (e.g., PKD1; Section 1.2.1; Ali et al., 2019; Prior et al., 2019).
Wherever phenotype numbers of the Online Mendelian Inheritance in Man® (OMIM®)
catalog (see “Online resources”) are used, these are provided in brackets; these are called
“MIM phenotype number” and also abbreviated as “[MIM XXXXXX]” throughout the
thesis. Finally, as only that individual of a family, in which genetic studies had been
started, was included in the study cohort of this thesis (i.e., not other relatives), the
individuals of the study cohort are referred to as “index patients” or “index cases,” or
simply “patients” or “cases.”
Hereditary kidney diseases comprise clinically and genetically heterogeneous conditions
affecting renal compartments such as the glomerulus (e.g., Alport syndrome [AS],
hereditary focal segmental glomerulosclerosis/steroid-resistant nephrotic syndrome
[FSGS/SRNS]) and tubuli (e.g., Bartter syndrome), altering embryonic development of
the kidney (e.g., congenital anomalies of the kidney and urinary tract [CAKUT]),
disturbing renal structure (e.g., ciliopathies), or occurring as part of metabolic (e.g., Fabry
disease) and mitochondrial disorders (e.g., mitochondrial encephalomyopathy, lactic
acidosis, and stroke-like episodes [MELAS]; Devuyst et al., 2014; Mehta & Jim, 2017;
Seidowsky et al., 2013). Furthermore, renal anomalies can be a frequent feature of
chromosomal aberrations, for example, monosomy X (Turner syndrome) or trisomy 21
(Down syndrome), which are not focused on in this thesis (Mehta & Jim, 2017).
In the following sections, an overview of hereditary nephropathies is presented:
Autosomal dominant tubulointerstitial kidney disease (ADTKD), AS, CAKUT,
ciliopathies, FSGS/SRNS, VACTERL/VATER (V – Vertebral anomalies, A – Anorectal
Introduction
27
malformations, C – Cardiovascular anomalies, T – Tracheoesophageal fistula, E –
Esophageal atresia, R – Renal and/or radial anomalies, L – Limb defects), and other
hereditary kidney diseases not fitting the aforementioned entities (tubulopathies, inherited
metabolic disorders, mitochondrial disorders, atypical hemolytic uremic syndrome).
These disease groups are used analogously to describe the ES study cohort in the results
section of the thesis.
(ADTKD)
ADTKD is an umbrella term for several rare hereditary kidney diseases that feature a
slow, progressive loss of renal function (end-stage renal disease [ESRD] in adulthood,
mean age 45 years) with unremarkable urinary sediment (marginal hematuria or
proteinuria). As ADTKD is a late-onset disease, reproduction is preserved, and pedigree
analysis can reveal affected individuals of both sexes in each generation compatible with
an autosomal dominant inheritance pattern. Renal biopsy can show unspecific fibrosis of
the tubular interstitium (hence tubulointerstitial kidney disease) and is not diagnostic of
disease. Renal cysts are prevalent in some patients but do not lead to enlarged kidneys, in
contrast to autosomal dominant polycystic kidney disease (ADPKD; Section 1.1.4). The
expressivity of disease can be highly variable within and across families. Treatment is
symptomatic, and kidney transplantation is curative, as there…

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